Methods and compositions for treating allergic reactions

ABSTRACT

Methods and compositions for treating allergic reactions, including cutaneous, ocular, nasal and Bronchial allergic disease, are disclosed. Interleukin-1 and Tumor Necrosis Factor receptors, and analogues thereof, are employed which bind the respective effector competitively and thereby suppress allergic reactions.

RELATED APPLICATION DATA

This application is a 371 of PCT/US92/08775, filed Oct. 14, 1992, whichis a continuation of U.S. patent application Ser. No. 07/776,624, filedOct. 15, 1991 and now abandoned.

TECHNICAL FIELD

The present invention relates to methods and compositions for treatingallergic reactions, and, more particularly, for treating bronchialasthma, rhinitis, rhinoconjunctivitis, conjunctivitis, and dermatitis.

BACKGROUND OF THE INVENTION

An allergic reaction is any abnormal or altered reaction to an antigen(or "allergen"). Typically such a reaction is characterized byhypersensitivity of the body to specific substances, whether protein,lipid or carbohydrate in nature. Allergic reactions may be local, e.g.contact dermatitis, or systemic, e.g. anaphylaxis.

Among allergic diseases, bronchial asthma is one of the mostsignificant. In most urban hospitals, it is the leading cause ofadmission of children. Current medical practice accepts asthma inafflicted individuals to be an unavoidable, incurable illness. Whilesuppression of symptoms is achieved to a degree sufficient to avoiddeath, urgent medical visits, disturbed sleep, and days lost from workare typically unavoidable.

The disease is generally associated with dyspnea, wheezing, and cough,as well as reversible airway obstruction and airway hyperreactivity tononspecific stimuli. These responses have been observed in two phases,early and late (Lemanske, Jr., R. F. and M. A. Kaliner, In: Allergy,Principles & Practice (3rd Ed.) pp. 224-246 (1988). See also Kaliner, M.A., Hosp. Prac. 22:73 (1987); Larsen, G., Hosp. Prac. 23:113 (1987)).

Inhalation of allergens by sensitized subjects typically results in anearly phase response characterized by bronchoconstriction within 10minutes of inhalation, reaching a maximum within 1 to 2 hours. In somesubjects, the airway narrowing recurs after 3 to 4 hours (i.e. a latephase response), reaching a maximum during the next few hours (O'Byrne,P. M. et al., Am. Rev. Respir. Dis. 136:740 (1987)). This late phasereaction is thought to be due to the cellular phase of inflammation(Hargreave, F. E. et al., Eur. J. Respir. Dis. 69(Suppl 147):16 (1986);O'Byrne, P. M., Chest 90:575 (1986); Dolovich, J. et al., J. AllergyClin. Immunol. 83(Suppl):521 (1987)).

No complete, long-lasting remissions of asthma have been described inresponse to any existing therapeutic strategies. For example,systemically administered glucocorticosteroids are potentantiasthmatics; however, the symptoms of the disease are onlytemporarily suppressed and this is at the cost of well-known sideeffects, including osteoporosis, weight gain, hypertension, and diabetes(Barnes, P. J., New Eng. J. Med. 321:1517 (1989)). Inhaled steroidtherapy also has complications (See Toogood, J. H., J. Allergy Clin.Immunol. 83(Suppl):528 (1987)). Low-dose methotrexate has been offeredas a substitute to steroids, particularly for patients for whom theside-effects of steroids are the most devastating (Mullarkey, M. F., NewEng. J. Med. 318:603 (1988)). However, methotrexate, while frequentlysubstituting for toxic doses of corticosteroids, has significantinherent toxicity. Furthermore, it does not eliminate the need forperiodic corticosteroids.

There is a great need for new approaches to treatment of allergicdisease. Specifically, there is a need for therapy that produceslong-lasting anti-inflammatory effect without harm to the patient.

DISCLOSURE OF THE INVENTION

The present invention relates to methods and compositions for treatingallergic reactions, including cutaneous, ocular, nasal, gastrointestinaland bronchial allergic disease.

In accordance with the present invention, at least one member selectedfrom the group consisting of Interleukin-1 (IL-1) receptors, TumorNecrosis Factor (TNF) receptors, and receptor analogues thereof whichbind the respective effector, is selectively employed to treat allergicreactions. The present treatment is expected to have many of thebeneficial effects of corticosteroids--however, without the toxicityassociated with these agents.

One aspect of the present invention contemplates using soluble IL-1receptors to treat inflammation in tissues. In certain embodiments, thismethod comprises contacting inflamed tissue with a therapeuticpreparation comprising soluble IL-1 receptors. In other embodiments, theinflamed tissue is skin and the inflammation is contact dermatitis,urticaria, angioedema or atopic dermatitis. In additional embodiments,the ocular tissue is inflamed and the inflammation is allergicconjunctivitis. In still other embodiments, the nasal tissue is inflamedand the inflammation is rhinitis. In yet additional embodiments, thelung tissue is inflamed and the inflammation is bronchial asthma.

The present invention also contemplates using such receptors or receptoranalogues in combination with other pharmaceuticals (e.g.corticosteroids) to treat inflammation in tissues. In certainembodiments, this method comprises contacting inflamed tissue with atherapeutic preparation comprising a mixture of such receptors andpharmaceuticals such as corticosteroids (e.g. prednisone) andantihistamines.

In other embodiments, the present method comprises contacting inflamedtissue with a therapeutic preparation comprising, in combination, bothTNF and IL-1 receptors.

The present invention also contemplates using an allergic assay toscreen for anti-allergic drugs. In one embodiment, the present inventioncomprises using a skin test to measure anti-inflammatory characteristicsof pharmaceuticals.

Therapeutic compositions containing such receptors and analogues thereofare also provided in accordance with the present invention.

BRIEF DESCRIPTION OF THE DRAWING

The single FIGURE schematically depicts exogenous receptors (shaded)competitively binding effectors (linked beads) to inhibit the binding ofthe effectors to endogenous receptors in a cell membrane.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to methods and compositions for treatingallergic reactions, and particularly, without limitation, bronchialasthma, rhinoconjunctivitis, conjunctivitis, dermatitis, urticaria,chronic bronchitis, allergic and non-allergic rhinitis and inflammatorylung disease. In accordance with the present invention, a therapeuticcomposition comprising at least one member selected from the groupconsisting of Interleukin-1 (IL-1) receptors, Tumor Necrosis Factor(TNF) receptors, and receptor analogues thereof which bind therespective effector, is applied exogenously to inflamed tissues. Thepresent invention contemplates the use of IL-1 receptors, TNF receptors,analogues thereof which bind the respective effector or combinations andmixtures thereof, in a therapeutic preparation.

It is presently considered desirable that the receptors and theiranalogues will be soluble in a medium appropriate to the particularapplication contemplated. As used herein, the term "soluble" shall meansufficient solubility in the selected medium so that the receptors arecapable of migrating to a position wherein they are able to bind withthe endogenous effector, unless a contrary meaning is clear form thecontext in which the term is used.

As used herein, the term "effector" shall mean IL-1 and/or TNFinterchangeably, unless a contrary meaning is clear form the context inwhich the term is used.

As used herein, the term "substantial" shall mean an amount sufficientto cause a detectable therapeutic effect, unless a contrary meaning isclear form the context in which the term is used.

IL-1 and IL-1 Receptors

Interleukin-1α (IL-1α) and Interleukin-1β (IL-1β) are distantly relatedpolypeptide hormones which play a central role in the regulation ofimmune and inflammatory responses (See Cerretti et al., U.S. Pat. Nos.4,894,333 and 4,879,374, each hereby incorporated by reference). Thesetwo proteins were originally both classified as IL-1, based on a sharedlymphocyte activation factor activity and a common major cellularsource, i.e. activated macrophages. As information has accumulated fromstudies using purified natural and recombinant IL-1 molecules, it hasbecome clear that IL-1α and IL-1β each mediate most, if not all, of thewide range of activities previously ascribed to IL-1. The basis for thisnearly identical spectrum of biological activities is thought to be thata single class of plasma membrane IL-1 receptors bind both IL-1α andIL-1β.

The existence of IL-1 plasma membrane receptors is now well-established.While original structural characterizations of the IL-1 receptor werelimited to estimates of the molecular weight of this protein by gelfiltration, by SDS-PAGE analysis of covalent complexes formed bychemical cross-linking between the receptor and ¹²⁵ I-IL-1 molecules,and by immunoprecipitation of labeled surface proteins, one of thereceptors has now been cloned and expressed in high yield (See Dower,U.S. Pat. No. 4,968,607 assigned to Immunex Corporation, herebyincorporated by reference).

TNF and TNF Receptors

Tumor Necrosis Factor (TNF-α) plays a critical role in the developmentof acute pulmonary failure and injury. When released into the lung,TNF-α has devastating effects, causing rapid and diffuse tissue injury.This is presumably a direct result of its known effects on endothelialcells and granulocytes, as well as its induction of other mediators suchas IL-1, prostaglandins, and platelet-activating factor. When TNF-α isinduced in the lungs of animals by the inhalation of endotoxin, airwaysdevelop a pattern of reactivity that is characteristic of bronchialasthma (See Pauwels, R. A. et al., Am. Rev. Resp. Dis. 141:540 (March1990)).

The use of blocking antibodies has made it possible to ablate the toxicaction of TNF-α. Baboons can be protected from a lethal intravenousdoses of E. coli organisms by prior administration of monoclonalanti-TNF-α(ab') fragments (See Tracey, N. J., Nature 330:662 (1987)).

Tumor Necrosis Factor-α and TNF-β receptors have been isolated and DNAsequences encoding these secretory proteins have been described (SeeSmith et al., European Patent Application No. 90309875.4 (PublicationNo. 0418014A1), assigned to Immunex Corporation, hereby incorporated byreference; See also U.S. patent application Ser. Nos. 07/405,370,07/421,417, and 07/523,635, hereby incorporated by reference).

Receptor Analogues

The present invention also contemplates the use of receptor analogues,and in particular IL-1 receptor analogues and TNF receptor analogues, astherapeutic agents. IL-1 receptor analogues are those compounds whichact in an analogous manner to competitively bind IL-1 and inhibit thebinding of IL-1 to endogenous IL-1 receptors. An example of such ananalogue is described in European Patent Application No. 343684, herebyincorporated by reference. In that case, the analogue is a polypeptideinhibitor of Interleukin-1. See also U.S. patent application Ser. Nos.07/199,915, 07/238,171, 07/248,521, and 07/266,531, each herebyincorporated by reference.

Such analogues which fall within the scope of the invention also includetruncated molecules, and molecules with amino acid additions,substitutions and deletions, wherein regions of the receptor moleculenot required for effector binding have been altered or deleted.

The analogues of the present invention share as a common, feature theability to competitively bind the respective effector to a degreesufficient to display a therapeutic effect when used in the practice ofthe present invention.

The Use of IL-1 Receptors and TNF Receptors

Since the IL-1 proteins are primary regulators of immunologicalresponses, some investigation has been made into the ability of IL-1receptors to modify immune responses. For example, Fanslow, W. C. etal., Science 248:739 (1990) describes the regulation of alloreactivityin vivo by a soluble form of the IL-1 receptor. They found that systemicadministration of the receptor prolonged the survival of heartallografts. In addition, Jacobs, C. A. et al., J. Immunol. 146:2983(1991) describes the use of soluble IL-1 receptor to suppressexperimental autoimmune encephalomyelitis. These researchers found thatinterperitoneal administration of the receptor reduced the severity ofthe disease.

In contrast to these previous uses of IL-1 receptor, the presentinvention contemplates the topical use of IL-1 receptor on inflamedtissue. In particular, the present invention contemplates the topical,as well as the parenteral use of IL-1 receptor to suppress the early andlate phase response in allergic reactions, including the late phaseresponse in bronchial asthma.

Similarly, the present invention contemplates the topical use of TNFreceptor on inflamed tissue. In particular, the present inventioncontemplates the topical, as well as the parenteral use of soluble TNFreceptor to suppress the early and late phase response in allergicreactions, including the late phase response in bronchial asthma.

While the benefits conveyed by treatment according to the presentinvention are not dependent on a precise understanding of themechanism(s) by which the subject receptors achieve a therapeuticresult, it is believed that the suppression of allergic responses isaccomplished by competitive binding of the exogenously supplied,receptors and/or analogues thereof to the respective effector, therebyinhibiting the binding of the effector to endogenous receptors on thetissue, as schematically portrayed in the Figure.

In the manner thus illustrated, IL-1 receptors or TNF receptors suppliedexogenously are expected to competitively bind to IL-1 or TNF,respectively, thereby inhibiting the binding of the effector to theendogenous receptor.

In practicing the method of the present invention, the therapeuticpreparation will be administered to a host in need of anti-allergictreatment at a therapeutically effective dosage level. The lowesteffective dosage levels can be determined routinely by initiatingtreatment at higher dosage levels and reducing the dosage level untilrelief from allergic reaction is no longer obtained. Generally,therapeutic dosage levels will range from about 0.01-100 μg/kg of hostbody weight.

Therapeutic Preparations and Combinations

The present invention contemplates using therapeutic compositions of thepresent receptors or analogues thereof to treat inflammation in tissues,as well as therapeutic preparations comprising, in combination, both TNFand IL-1 receptors. Furthermore, the present invention also contemplatesusing IL-1 receptors, TNF receptors, receptor analogues, andcombinations thereof in combination with corticosteroids or otherantiinflamatory drugs or molecules in a therapeutic preparation to treatinflammation in tissues.

Where combinations are contemplated, it is not intended that the presentinvention be limited by the particular nature of the combination. Thepresent invention contemplates combinations as simple mixtures as wellas chemical hybrids. One example of the latter is where the receptor iscovalently linked to a pharmaceutical such as a corticosteroid, or wheretwo receptor types are joined. For example, covalent binding of thedistinct chemical moieties can be accomplished by any one of manycommercially available cross-linking compounds. Further examples of suchchemical hybrids include combinations of the receptors together or withother biologically active or inert molecules prepared to utilize theeffects of IL-1 receptor and/or TNF receptor. Such hybrid or fusionmolecules can be constructed using the techniques of geneticengineering. Similar such molecules have been created by several methodsutilizing promoter genes (See, e.g., Feng, G. et al., Science 241:1501(1988)).

It is not intended that the present invention be limited by theparticular nature of the therapeutic preparation. For example, suchcompositions can be provided together with physiologically tolerableliquid, gel or solid carriers, diluents, adjuvants and excipients.

These therapeutic preparations can be administered to mammals forveterinary use, such as with domestic animals, and clinical use inhumans in a manner similar to other therapeutic agents. In general, thedosage required for therapeutic efficacy will vary according to the typeof use and mode of administration, as well as the particularizedrequirements of individual hosts.

Such compositions are typically prepared as sprays (e.g. intranasalaerosols) for topical use. However, they may also be prepared either asliquid solutions or suspensions, or in solid forms including respirableand nonrespirable dry powders. Oral formulations (e.g. forgastrointestinal inflammation) usually include such normally employedadditives such as binders, fillers, carriers, preservatives, stabilizingagents, emulsifiers, buffers and excipients as, for example,pharmaceutical grades of mannitol, lactose, starch, magnesium stearate,sodium saccharin, cellulose, magnesium carbonate, and the like. Thesecompositions take the form of solutions, suspensions, tablets, pills,capsules, sustained release formulations, or powders, and typicallycontain 1%-95% of active ingredient, preferably 2%-70%.

The compositions are also prepared as injectables, either as liquidsolutions or suspensions; solid forms suitable for solution in, orsuspension in, liquid prior to injection may also be prepared.

The receptors of the present invention are often mixed with diluents orexcipients which are physiologically tolerable and compatible. Suitablediluents and excipients are, for example, water, saline, dextrose,glycerol, or the like, and combinations thereof. In addition, if desiredthe compositions may contain minor amounts of auxiliary substances suchas wetting or emulsifying agents, stabilizing or pH buffering agents.

Additional formulations which are suitable for other modes ofadministration, such as topical administration, include salves,tinctures, creams, lotions, and, in some cases, suppositories. Forsalves and creams, traditional binders, carriers and excipients mayinclude, for example, polyalkylene glycols or triglycerides.

The following examples serve to illustrate certain preferred embodimentsand aspects of the present invention and are not to be construed aslimiting the scope thereof.

EXPERIMENTAL

In the experimental disclosure which follows, the followingabbreviations apply: eq (equivalents); M (Molar); μM (micromolar); N(Normal); mol (moles); mmol (millimoles); μmol (micromoles); nmol(nanomoles); kg (kilograms); gm (grams); mg (milligrams); μg(micrograms); L (liters); ml (milliliters); μl (microliters); cm(centimeters); mm (millimeters); μm (micrometers); nm (nanometers); and°C. (degrees Centigrade).

Example 1

This example describes the use of soluble IL-1 receptor to reduce thecutaneous allergic reactions following the intradermal administration ofantigen.

Early and late phase responses have been observed following bronchialchallenge with such antigens as ragweed pollen and house dust.Importantly, these responses in lung tissue correspond in time to theearly and late phase reactions in skin following intradermal challengewith similar antigens (See Dolovich, J. and D. C. Little, J. AllergyClin. Immunol. 49:43 (1972); See also Solley, G. O.. et al., J. Clin.Invest. 58:408 (1976)). Skin reactivity to allergen, especially the LatePhase Reaction (LPR) skin test is believed to be predictive of eventsoccurring in the lungs of asthmatics.

The LPR skin test is performed by intradermally injecting subjects onthe forearm with test solution. The test solution contains thechallenging antigen. Controls receive test solution without challengingantigen. The injection site is thereafter examined at intervals up to 96hours. The diameters of the reactions are measured in two perpendiculardirections and the characteristics are noted at different times.

Most subjects exhibit a dual reaction, the LPR beginning at the 4 hourpoint and reaching a peak at the 8-12 hour point. The LPR graduallysubsides over a 24 hour period. At the peak, the injection site ischaracterized by erythema, warmth, edema, pruritus and/or tenderness.The LPR is more extensive in area and produces greater discomfort thenthe early phase reaction.

In this example, the LPR skin test is performed on individuals; all havepreviously had a dual response (early and late) to intradermal challengewith antigen as described above. Dust mite antigen is used (D.pteronyssinus; Allergy Laboratories of Ohio; 30,000 AU/ml). The solubleIL-1 receptor (Immunex Corp.; Seattle, Wash.) stock concentration is 10mg/ml. Eight test solutions are prepared for intradermal injections (0.1ml each) using TB syringes and disposable needles. Four intradermal testsites are used on each forearm.

Two test solutions are controls. Histamine phosphate (Allermed Labs; 1.8mg/ml) is used as a positive control, and saline is used as a negativecontrol. The six remaining test solutions all contain dust mite antigen;three contain IL-1 receptor (final concentration 1.65 mg/ml) while theother three contain saline. Three concentrations of the antigen areevaluated (final concentrations: 1:500,000, 1:200,000, and 1:100,000).

The area of the wheal reaction is marked on the skin with a ball-pointpen. Clear adhesive tape is then applied to the marked skin. The tapewith the pen markings is removed from the skin and taped on paper with0.1 mm squares. The wheal reactions are then calculated by counting thesquares within the pen-marked area.

At twenty minutes post injection, the control allergen injection (i.e.containing no receptor) at the lowest concentration produces a whealmore than half the area of the histamine reaction after subtracting theresponse to the diluent control. By contrast, the wheal produced byinjections containing IL-1 receptor are

At two hours the early phase subsides. The LPR peaks between eight and13 hours. At its peak, the control allergen injection (i.e. containingno receptor) at the lowest concentration produces a wheal larger thanthat observed at 20 minutes, while the wheal produced by injectionscontaining IL-1 receptor are reduced by at least 50 percent.

The results of this example, while specific for IL-1 receptors,nonetheless shows the general applicability of using an allergic assayto screen for anti-allergic drugs. Indeed, this skin test is appropriateto measure anti-inflammatory characteristics of any pharmaceutical.

If desired, a pathologic assessment can be made of the allergic reactionin addition to measuring the wheal visually in the simple skin test. Ateight hours a punch biopsy can be taken at one or more of the allergensites on each forearm. Standard hematoxylin and eosin staining can beperformed on formalin-fixed, paraffin-embedded sections. The assessmentis made according to the pattern and type of cellular infiltration atthe biopsy sites. The sites can be graded on a 0 to 3 scale (none, mild,moderate or severe on the basis of presence and extent of i)perivasfular infiltrate, ii) interstitial infiltrate and edema, and iii)leukocytoclasis. Particular note can be taken with regard to thepresence of lymphocytes, eosinophils, macrophages, granulocytes and anyother cellular element which is increased in number or unique to normalskin.

Example 2

This example describes the use of soluble IL-1 receptor to reduce theLPR following conjunctival provocation with antigen.

Ocular involvement is common in allergic conditions. It can be theresult of systemic allergic symptoms or, indeed, the main focus ofallergic disease.

With respect to specific ocular allergies, allergic rhinoconjunctivitis,atopic keratoconjunctivitis, vernal conjunctivitis, giant papillaryconjunctivitis, and contact allergy have been identified as the primarytypes. The most commonly seen form of ocular allergy is the red itchyeyes that accompany allergic rhinitis during the allergy season.

Usually ocular symptoms are overshadowed by nasal or respiratorysymptoms. However, in some cases the ocular symptoms predominate.Patients typically complain of red, swollen, itchy eyes, and scantmucous discharge. Itching is an important symptom for ocular allergysince most patients with allergy have itching, and very few other ocularconditions are associated with itching. Other common ocular reactionsinclude giant papillary conjunctivitis (associated with contact lenses)and contact allergy (caused frequently by soaps, shampoos, and eyemakeup).

To study ocular involvement, allergists have developed protocols tomeasure conjunctival allergic response. Conjunctival provocation testsin allergic individuals have been used to confirm the diagnosis ofallergy, study the physiologic changes accompanying the allergicreaction, sample the cells and mediators of the allergic response, andevaluate anti-inflammatory therapy.

The conjunctival provocation test (CPT) is a good way of determining thepresence or absence of allergy. This is particularly true when a skintest is negative or equivocal. Moreover, the CPT has been found to besafe; the cornea is not affected, and patient discomfort is mild andtransient.

Eosinophilia is normally absent in conjunctival scrapings taken fromnonallergic individuals. The presence of conjunctival eosinophilia isconsidered to be a diagnostic indicator of allergic conjunctivitis, andthe severity of the disease appears to correlate with the level of majorbasic protein in tears.

The tear fluid can also be sampled to evaluate the mediators of ocularinflammation. Tear IgE levels have been measured in allergic patientsand in general, there is a correlation between the tear and serum IgElevels. This correlation exists when serum IgE is greater than 100 IU/mland tear IgE is greater than 4 IU/ml.

To evaluate the severity of the response, the eye can be examined with astrong flashlight, or if available, a slit-lamp microscope. The allergicconjunctive appears inflamed and edematous. Rather than intense rednessand prominence of blood vessels, the conjunctiva has a pinkish or milkyappearance.

In the present example, inhibition of the ocular symptoms in the LPR isexamined; the presence of inflammatory cells in the tear film iscorrelated with the occurrence of ocular symptoms in the LPR timeperiod.

Ten ryegrass-sensitive patients with hay fever conjunctivitis and tennonallergic subjects without ocular disease are challenged by weeklytopical administration of ryegrass allergen for 4 weeks with 10 μl offour different allergen doses (10,000, 32,000, 100,000, and 320,000BU/ml of ryegrass allergen) (Pharmalgen, Pharmacia Diagnostics AB,Uppsala, Sweden). Albumin diluent (Pharmacia), is used to dilute theallergen. Five of the patients and five of the nonallergic subjects aregiven allergen premixed with IL-1 receptor. The other five patients andsubjects are given allergen without IL-1 receptor. Importantly, theallergic patients and control subjects have no clinical symptoms beforeocular provocation.

The CPT involves introducing allergen to the lower conjunctival fornixof one eye by applying 10 μl of different dilutions of ryegrass inalbumin prepared at the time of testing. The concentration used were10,000, 32,000, 100,000, and 320,000 BU/ml. The other eye was used as acontrol; at the same time the control eye received 10 μl of albumin.

The challenge solution is administered to the contralateral eye in bothpatients and control subjects. Clinical conjunctival evaluation withtear-fluid cytology is assessed in both eyes before administration ofallergen or buffer 20 minutes, one hour, and six hours after challenge.In both the allergic patients and control subjects, the following ocularsymptoms, both subjective and objective, are evaluated by a physician:hyperemia, edema, tearing, and itching. Each symptom is scored from 0 to4+ and graded as follows: 0--absent; 1+--mild; 2+--moderate; 3+--severe;and 4+--very severe. The sum of all scores (maximal clinical score, 16)obtained from each item represented the intensity of the clinicalreaction at each time point.

A microcapillary tube (Sigma Chemical Co., St. Louis, Mo.) is used tocollect 2 μl of tears from the inner canthus without touching the ocularsurface. Such collection of tears is a noninvasive technique whichallows repeated sampling without conjunctival trauma. Tears are spreadon a glass slide, air-dried, and stained with Wright-Gemsa (Diff-Quick,Baxter Healthcare Corp., Gibbstown, N.J.). All identifiable cells oneach slide are counted at original magnification ×1000 using lightmicroscopy. Five types of cells are scored: epithelial cells,neutrophils, eosinophils, lymphocytes, and monocytes. All participantsare first examined prior to provocation to assure that there is nosignificant difference in the baseline levels of inflammatory cells inthe tear fluid of allergic patients compared with levels of the controlsubjects.

Following provocation, all allergic subjects receiving allergen withoutIL-1 receptor exhibit evidence of an immediate hypersensitivity ocularreaction with all allergen doses; allergic subjects receiving allergentogether with IL-1 receptor, exhibit a significantly reduced response.The conjunctival response in allergen-challenged (no receptor) eyes ofthe allergic patients is also statistically significant compared withthe albumin diluent-treated eyes. A significant number of neutrophilsare detected in eyes challenged with 320,000 BU/ml of ryegrass allergen(no receptor) compared with the those receiving this dose together withIL-1 receptor.

The highest allergen dose causes the recruitment of a significant numberof eosinophils and lymphocytes to the tear fluid 6 hours afterprovocation (i.e. in late phase) in those eyes challenged with allergenwithout receptor. This increase in eosinophils in the tear fluid is notseen in allergic patients receiving allergen with receptor.

Example 3

This example describes the use of soluble IL-1 receptor to reduce theLPR in nasal tissue following inhalation of antigen.

Patients with allergic rhinitis often have immediate symptoms afterantigen challenge (the early phase response), followed several hourslater by a recurrence of symptoms (the late-phase response). Thisexample involves a controlled study of asymptomatic subjects in thepollen-free winter months.

Sixteen patients with seasonal allergic rhinitis due to grass or ragweedpollens are selected. All subjects have a positive intradermal skin testto 10 PNU (protein nitrogen units) or less of antigen extract, and allhave previously had a dual response (early and late) to nasal challengewith antigen.

Ragweed and mixed-grass pollen extracts (timothy, orchard, June andmeadow grass in a ratio of 3:2:3:2) are purchased from GreerLaboratories (Lenoir, N.C.); lactated Ringer's solution andoxymetazoline hydrochloride (Afrin, Schering, Kenilworth, N.J.) arepurchased from the hospital pharmacy.

At the time of the challenge, oxymetazoline hydrochloride is sprayedinto the nose (two sprays per nostril) of all patients to preventmucosal congestion, which would interfere with the collection of nasalsecretions. It has been shown previously that this dose of oxymetazolinedoes not affect histamine release during the early reaction to antigen.

Eight of the sixteen patients receive four prechallenge nasal lavageswith IL-1 receptor diluted in buffer; the remaining ten receive lavageswith buffer only. Thereafter, challenges with 1000 PNU of antigen areundertaken.

The patients maintain a symptom score sheet during the challengeprocedure. In addition to the number of sneezes, a six-point scale from0 to 5 (with 0 equal to no symptoms and 5 equal to severe symptoms) isused to assess nasal secretion, blockage, and itching. The degree ofblockage is, of course, underestimated on those score sheets because ofthe pretreatment with oxymetazoline hydrochloride. The presence orabsence of symptoms correlates with the presence or absence of mediatorsduring the late reaction.

From a comparison of those patients receiving prechallenge treatmentwith IL-1 receptor and those receiving only buffer, it is clear thatpretreatment with IL-1 receptor inhibits both the symptoms and therelease of histamine and other inflammatory mediators during not onlythe late and rechallenge reactions to nasal challenge with antigen butalso the early response.

Example 4

This example describes the use of soluble IL-1 receptor to reduce thelate phase reaction (LPR) in lung tissue following inhalation ofallergen.

Airway hyperactivity can be induced or worsened by antigen inhalation,exposure to some irritating chemicals, and by respiratory tractinfections. The degree of reactivity is directly correlated with thenumber of mast cells and eosinophils detected by lavage.

In this example, twenty patients with documented allergic bronchialasthma participate. Allergen inhalation is performed by inhaling dustmite extract (see Example 1, above) at 15 minute intervals using aWiesbadnener Doppelspray (8 L/min air flow, nebulizer outputapproximately 0.2 ml/min). Ten patients received the allergen premixedwith IL-1 receptor; the other ten patients received the allergen alone.

Bronchoalveolar lavage performed 6 to 48 hours after inhalation ofallergen alone shows increased numbers of eosinophils, mast cells, anddesquamated epithelial cells. Eosinophil granule major basic proteinlevels are markedly elevated in the lavage fluid of these asthmaticpatients. Peripheral blood eosinophil counts decrease during late phaseresponses to antigen challenge at the time eosinophil levels in thepulmonary tissues increases, presumably because of margination andemigration in the lungs.

Bronchoalveolar lavage after inhalation of allergen with IL-1 receptorshows suppression of eosinophil emigration in the lungs. The histologicdata is consistent with the concept that activation of mast cells,infiltration of the tissues by eosinophils and other inflammatory cells,and tissue damage as well as dysfunction induced by inhalation ofallergen is markedly suppressed by the presence of IL-1 receptor.

Thus it has been shown that the present invention provides beneficialmethods and compositions for treating allergic reactions, including,without limitation, bronchial asthma, rhinoconjunctivitis,conjunctivitis, dermatitis, urticaria, chronic bronchitis, allergic andnon-allergic rhinitis and inflammatory lung disease.

All publications and patent applications cited in this specification areherein incorporated by reference as if each individual publication orpatent application were specifically and individually indicated to beincorporated by reference.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity andunderstanding, it will be apparent to those of ordinary skill in the artin light of the teaching of this invention that certain changes andmodifications may be made thereto without departing from the spirit orscope of the appended claims.

We claim:
 1. A method for treating tissue subject to reactionscharacterized as having late phase inflammatory responses comprisingcontacting the inflamed tissue with a preparation comprising at leastone member selected from the group consisting of IL-1 receptors, TNFreceptors, and receptor analogues thereof which are capable of bindingeither IL-1 or TNF.
 2. The method of claim 1 wherein the inflammatoryreaction is selected from the group consisting of cutaneous, ocular,nasal, gastrointestinal and bronchial reactions.
 3. The method of claim2 wherein the inflammatory reaction is selected from the groupconsisting of contact dermatitis, atopic dermatitis, urticaria,conjunctivitis, rhinitis, rhinoconjunctivitis, systemic anaphylaxis,asthma, hay fever, chronic bronchitis and inflammatory lung disease. 4.The method of claim 1 wherein a substantial proportion of the receptorsor analogues contained in the preparation are soluble.
 5. The method ofclaim 1 wherein the preparation comprises a mixture of IL-1 receptorsand TNF receptors.
 6. The method of claim 5 wherein at least a portionof said TNF receptors are covalently bound to said IL-1 receptors. 7.The method of claim 1 wherein the preparation comprises analogues ofeither IL-1 receptors or TNF receptors.
 8. The method of claim 7 whereinat least a portion of said receptor analogues are hybrid fusionmolecules comprising a TNF receptor moiety and an IL-1 receptor moiety.9. The method of claim 1 wherein the preparation further comprises atherapeutically effective amount of at least one corticosteroid.
 10. Themethod of claim 9 wherein at least a portion of said corticosteroids arecovalently bound to said receptors.